Microscopes magnify objects or samples, which can be stationary and moving. One type of microscope is a confocal microscope, which uses a very small spot, or pinhole, of light to make its image of the target. Typically, the spot is scanned across the target in a pointwise, digital fashion and the image is made by combining the points of return light emanating from the target (the return light can be, for example, reflected light, fluorescent light, or an exotic form of light such as a Raman spectrum, and can be found in any desirable region of the electro-magnetic spectrum, such as ultraviolet (UV) light, blue light, visible light, near-infrared (NIR) light and infrared (ER) light).
The confocal geometry of the illumination pinhole, the object, and the detection pinhole give a higher resolution image than a conventional widefield microscope. In some embodiments, confocal microscopy can improve the spatial resolution about 1 3 times. See, e.g., U.S. Pat. No. 5,587,332. Confocal microscopy also improves the “up and down” (i.e., z-axis or axial) resolution, which gives rise to an extremely useful optical sectioning capability; which means that images can be obtained at different depths, and thus 3-D images and volume reconstruction can be obtained.
In order to obtain the pointwise image, confocal microscopes can either move a specimen and keep the optics fixed in place, or they can keep the specimen fixed and move the light beam, for example by scanning the beam using special rotating aperture disks or other beam scanners. See U.S. Pat. No. 4,802,748, U.S. Pat. No. 5,067,805, U.S. Pat. No. 5,099,363, U.S. Pat. No. 5,162,941. Other confocal scanning systems have used a laser beam rastered with rotating mirrors to scan a specimen or a laser beam that scans a slit rather than a spot; such slit scanning increases imaging speed but slightly degrades resolution. See U.S. Pat. No. 5,587,332.
Confocal microscopes typically use a bulky design in which several large components—including a laser system as the light source, detection pinholes, x-y beam steering devices, and an optical detector—must be carefully maintained in precise alignment. In these systems, the specimen or target to be imaged is placed on a stage as in a conventional microscope. These limitations make the confocal microscope cumbersome, inflexible and inconvenient for imaging specimens which are not easily accessible or easily placed on a microscope stage. In other words, present confocal systems are designed for in vitro imaging of biological specimens in the lab instead of imaging tissues in the body, in vivo.
Several approaches have been proposed to permit in vivo imaging. See, e.g., T. Dabbs and M. Glass, “Fiber-optic confocal microscope: FOCON,” Applied Optics, vol. 31, pp 3030-3035, 1992; L. Giniunas, R. Juskatis, and S. V Shatalin, “Scanning fiber-optic microscope,” Electronic Letters, vol. 27, pp. 724-725, 1991; L. Giniunas, R. Juskatis, and S. V. Shatalin, “Endoscope with optical sectioning capability,” Applied Optics, vol 32, pp. 2883-2890, 1993; D. L. Dickensheets and G. S Kino, “Micromachined scanning confocal optical microscope,” Optics Letters, vol. 21, pp. 764-766, 1996; D. L. Dickensheets and G. S. Kino, “Miniature scanning confocal microscope,” U.S. Pat. No. 5,907,425 (continuation of 5,742,419), May 1999; A. F. Gmitro and D. Aziz, “Confocal microscopy through a fiber-optic imaging bundle,” Optics letters, vol. 18, pp. 565-567, 1993; Y. S. Sabharwal, A. R. Rouse, L. Donaldson, M. F. Hopkins, and A. F. Gmitro, “Slit-scanning confocal microendoscope for high-resolution in vivo imaging, Applied Optics, vol. 38, pp. 7133-7144, 1999; R. Juskaitis, T. Wilson, and I. F. Watson, “Confocal microscopy using optical fibre imaging bundles,” Proceedings of SPIE, vol. 2655, pp. 92-94, 1996; U.S. Pat. No. 5,587,832; PCI/CA98/00993, Publication No. WO 99/2.2.262. None of these systems provide as high a quality of image as could be desired for various aspects of microscopy.
Thus, there has gone unmet a need for improved microscopy systems, including confocal microscopy systems, wherein the systems can provide high quality images of desired targets in locations where the positioning of the target might not be carefully controlled, including in vivo targets. The present invention provides these and other advantages.